Sterols are abundant lipids in mammalian cells that maintain the structural integrity of the plasma membrane, form specialized signaling centers termed lipid rafts, and serve numerous regulatory roles as agonists and antagonists of transcription. The diverse functions of sterols, together with their structural complexity, suggest that novel sterols and biological roles remain to be identified for this lipid class. The initial goal of Core J will be to develop lipidomics methods to advance mechanistic understanding of sterol metabolism in the macrophage. Known sterols will be identified and quantitated by LC-MS methods in immortalized and primary macrophages subjected to lipopolysaccharide, cytokine, and phagocytic challenges, or treated with inhibitors of lipid biosynthesis. Novel sterols and other lipids will be identified in these experiments using an LC-MS based metabolomics approach in which comparisons are made between total lipid profiles derived from experimental and control cells. The structures of new lipids will be determined by combined LC-MS and GC-MS analyses, and cDNAs encoding their biosynthetic enzymes will be isolated by expression cloning. The second objective of Core J is to employ lipidomics to investigate the sterol complement of macrophages and tissues under normal and pathological conditions. Sterols will be extracted from cells and tissues isolated from different animal models of disease or diet-induced pathologies and then quantitated by LC-MS. The effects of drugs such as statins and glitazones on sterols levels will be determined in these experiments, and cross-talk between different lipid pathways will be assessed by the above LC-MS based metabolomics approach. Our third goal will be the development of lipid networks and maps from the data generated in the above studies. Sterol and other lipid profiles will be compared to gene expression data generated by microarray analyses to produce a global picture of the lipidome, including the biosynthetic enzymes involved, pathway interactions, and metabolite flows. Stable isotope methods using 13C-mevalonate as a precursor will be developed to provide a detailed understanding of sterol synthesis and turnover. The proposed research will provide new insight into the roles of sterols and other lipids in the macrophage, a cell type that is relevant to immunity, inflammation, and atherosclerosis.